Occurrence of poly-d(−)-3-hydroxyalkanoates in the genus Bacillus

A range of Bacillus strains were examined for their ability to accumulate poly-D(-)-3-hydroxyalkanoates (poly-HAKs) which are naturally occurring materials that are optically active, biodegradable thermoplastics. The organisms could produce poly-D(-)-3-hydroxybutyrate (poly-HB) up to 50% of cell dry weight. The content of poly-HB in the cells varied with the growth conditions. The addition of propionate or valerate in the culture resulted in a synthesis of poly-D(-)-3-hydroxyvalerate (poly-HV). All the strains tested had the ability to synthesize the co-polyester poly-HB-co-HV.     

Vergleichende Untersuchung der Proteinasen zweier Bacillus-Arten (Bacillus subtilis und Bacillus cereus)

Ohne ZusammenfassungTeil der Dissertation, Universität Frankfurt am Main 1958.     

Four-Iron (Sulfide) Ferredoxin from Bacillus polymyxa

Ferredoxin from Bacillus polymyxa contains (per mole) four non-heme iron residues, four acid-labile sulfide residues, and four cysteine residues. Its molecular weight is approximately 8,800, and it has an oxidation-reduction potential (E(m)) of -390 mv. It is active as an electron carrier in several ferredoxin-linked enzyme systems.     

Insertion mutagenesis in Bacillus subtilis (Marmur)

A method for obtaining mutations in Bacillus subtilis using integration into the chromosome of the plasmid pHV60 ligated to chromosomal DNA fragments was developed. Auxotrophic mutants acquire, in addition, chloramphenicol-resistance, due to insertion of appropriate plasmid determinants. Chromosomal localization was established and the properties of several mutants were studied.     

Bacteria-shaped Gymnoplasts (Protoplasts) of Bacillus subtilis

Addition of glucose to the medium in which Bacillus subtilis was grown lowered the pH and increased the amount of lysylphosphatidylglycerol relative to the phosphatidylglycerol content of the membrane fraction. This change in phospholipid composition was accompanied by changes in the shape and behavior of the gymnoplasts obtained by cell wall removal with lysozyme. These gymnoplasts appeared to retain most of their original cell shape and internal organization, often with preservation of the mesosomes. Cells harvested from neutral growth medium gave the usual spherical gymnoplasts. In a hypotonic medium, the spherical gymnoplasts lysed rapidly, whereas the rod-like gymnoplasts lost only part of their cell content while showing a tendency to preserve the original shape. This type of gymnoplast could not be produced from cells grown in neutral medium by simply raising the magnesium concentration. When this was done the gymnoplasts assumed bizarre shapes; they became compact and susceptible to the tonicity of the medium. Gymnoplasts or protoplasts, produced from bacilli exposed to low pH values, were found not to conform to the formulations on "protoplasts" given in 1958 by 13 authors. Cells exposed to a low environmental pH during growth seemed to possess a more rigid membrane structure than cells grown at neutral pH.     

Ca-binding to Bacillus licheniformis alpha-amylase (BLA)

Ca-induced renaturation of Bacillus licheniformis alpha-amylase in the presence of urea has been employed to determine the binding constants of the ion. The native enzyme is folded at 3M urea while the Ca-depleted protein is largely unfolded at this denaturant concentration. Refolding of the protein has been monitored by circular dichroism and the titration curves have been analyzed assuming a model of three independent binding sites. The stoichiometry has been taken from X-ray studies. The refolded protein exhibits a secondary structure that is similar but not identical to that of the native protein. The binding constants have been used to construct a phase diagram that illustrates the contribution of Ca-binding to the resistance against urea unfolding.     

Adaptation of Bacillus megatherium to terramycin (oxytetracycline)

B. megatherium cultures contain a few cells per hundred million which are able to grow in the presence of terramycin. Growth of the other cells is inhibited by the antibiotic with the result that the resistant cells overgrow the culture. The resistant cells are present in the culture, without contact with terramycin, and are able to grow in the presence of terramycin, without further modification. The resistant cells are mutants of sensitive cells.     

As(V) Reduction,As(III) Oxidation,and Cr(VI) Reduction by Multi-metal-resistant Bacillus subtilis,Bacillus safensis,and Bacillus cereus Species Isolated from Wastewater Treatment Plant

Industrial progress has resulted in threatening concentrations of toxic metals in various areas of the world. Bioremediation is an economical alternative to chemical methods. Bacteria resistant to As(III), As(V), Cr, Co, Cu, Cd, Hg, Ni, Pb, Se, and Zn were isolated from the wastewater treatment plant of Kasur, Pakistan. Highest resistance against all metals was exhibited by MX-1, MX-3, MX-4, and MX-5. The isolates possessed dual ability to oxidize as well as to reduce As. Highest As(V) reduction (454 µM) was exhibited by MX-1, while most As(III) oxidation was shown by MX-3 (170 µM). The isolates were also capable of reducing Cr(VI), and maximum Cr(VI) reduction (500 µM) was exhibited by MX-3. Transformation of DH5α with MX-1 plasmid showed that resistance genes for As(III), As(V), Cr, Cd, Se, Hg, and Ni were plasmid borne, while in case of MX-3, resistance genes for As(III), As(V), Co, Cu, Se, Pb, Zn, and Ni were present on plasmid. MX-1 and MX-3 were also positive for auxin production (37 and 32.96 µg ml^?1, respectively). MX-3 was also found to produce hydrogen cyanide (HCN) and solubilized phosphate. These isolates promoted plant (Vigna radiata) growth both in the presence and absence of the metals. MX-1, MX-3, and MX-5 were identified as Bacillus subtilis, Bacillus safensis, and Bacillus cereus through 16S rRNA gene sequencing, respectively. Such bacteria having multiple traits of resisting multiple metals, dual ability to oxidize/reduce As, and reduce Cr(VI) along with the ability to support plant growth are good tools for remediation of metal-contaminated sites and its cultivation.     

Effects of BCG (Bacillus Calmette–Guérin) Vaccines on Immune Responses in Mice

The effects of killed and living BCG on antibody production against hamster erythrocytes (HRBC) and the 2, 4, 6-trinitrophenyl (TNP) group were studied in SL mice. Killed and living BCG, each in doses of 0.008 mg, 0.08 mg, 0.8 mg and 8 mg per mouse, were intravenously inoculated 7 days prior to primary immunization with HRBC. Secondary immunization was carried out 28 days later with TNP-HRBC. Anti-HRBC and anti-TNP antibodies were estimated by a hemagglutination test. The results showed that pretreatment with killed or living BCG enhanced the antibody production against both HRBC and TNP. Comparing the effects of these two BCG preparations, it was noted that killed BCG augmented the anti-HRBC antibody production more effectively than living BCG. In regard to the anti-TNP antibody production, living BCG exhibited a greater augmenting effect than killed BCG. This difference in the modes of action of killed and living BCG was remarkable when two groups given 8 mg of killed and living BCG were compared. In addition, it was shown that living BCG at a dose as high as 8 mg was able to augment the anti-TNP antibody production, even in the absence of preceding immunization with HRBC.     

Regulation of alanine dehydrogenase in Bacillus (licheniformis)

Cell extracts of Bacillus licheniformis were found to contain nicotinamide adenine dinucleotide (NAD)-dependent l-alanine dehydrogenase (ADH) (l-alanine: NAD oxidoreductase, EC 1.4.1.1). High specific activities (3.5 to 6.0 IU/mg of protein) were found in extracts of cells throughout growth cycles only when l-alanine served as the primary source of carbon or carbon and nitrogen. Specific activities were minimal (0.02 to 0.04 IU/mg of protein) during growth on glucose, but increased at least sevenfold during the first 5 h of postlogarithmic-phase metabolism. Addition of 10 mM glucose to cultures during logarithmic-phase growth on l-alanine resulted in a rapid decrease in enzyme activity. Addition of 20 mM l-alanine to cells near the completion of log-phase growth on glucose resulted in a 20-fold increase in ADH specific activity during less than one cell generation. Extracts of postlogarithmic-phase cells cultured on glucose, malate, l-glutamate, or Casamino Acids contained intermediate levels of ADH activity. The enzyme was partially purified from crude extracts of B. licheniformis, and apparent kinetic constants were estimated. A role for ADH in the catabolism of l-alanine to pyruvate during vegetative growth on l-alanine and during sporulation of cells cultured on glucose is proposed on the basis of these experimental results.     

Isolation and purification of cell wall polysaccharide of Bacillus anthracis (Δ Sterne)

A polysaccharide fraction was isolated form sodium-dodecyl-sulfate (SDS) treated cell walls of Bacillus anthracis (delta Sterne) by hydrofluoric acid (HF) hydrolysis and ethanolic precipitation. The polysaccharide fraction was subsequently purified by several washings with absolute ethanol. Purity of the isolated polysaccharide was tested using the anthrone assay and amino acid analyzer. The molecular mass of the polysaccharide fraction as determined by gel filtration chromatography was about 12000 Da. Preliminary analyses of the polysaccharide was done using thin layer chromatography and amino acid analyzer, and results obtained from these analyses were further confirmed by gas liquid chromatography and 13C-NMR spectroscopy. Results showed that the polysaccharide moiety contained galactose, N-acetylglucosamine, and N-acetylmannosamine in an approximate molar ratio of 3:2:1. This moiety was devoid of muramic acid, alanine, diaminopimelic acid, glutamic acid, and lipid, thus indicating that the isolated polysaccharide was of pure quality.     

Phylogenetic Analysis of Bacillus subtilis Strains Applicable to Natto (Fermented Soybean) Production

Spore-forming Bacillus strains that produce extracellular poly-γ-glutamic acid were screened for their application to natto (fermented soybean food) fermentation. Among the 424 strains, including Bacillus subtilis and B. amyloliquefaciens, which we isolated from rice straw, 59 were capable of fermenting natto. Biotin auxotrophism was tightly linked to natto fermentation. A multilocus nucleotide sequence of six genes (rpoB, purH, gyrA, groEL, polC, and 16S rRNA) was used for phylogenetic analysis, and amplified fragment length polymorphism (AFLP) analysis was also conducted on the natto-fermenting strains. The ability to ferment natto was inferred from the two principal components of the AFLP banding pattern, and natto-fermenting strains formed a tight cluster within the B. subtilis subsp. subtilis group.     

Brownian dynamics simulation of the association of Bacillus amyloliquefaciens ribonuclease (barnase) and Bacillus intermedius ribonuclease (binase) with barstar

A comparative study of the association of two ribonucleases, barnase and binase, with the polypeptide inhibitor barstar has been performed by the Brownian dynamics simulation method. It was shown that the method adequately reproduced the dependence of the association rate on pH and ionic strength of solution and the influence of mutations of some ribonuclease amino acids. Two types of energetically favorable complexes of binase-barstar encounter were determined. In the type I complex, the amino acids of binase active center take part in the complex formation. In the second complex, the active center is free. It was supposed that the temporary binding of barstar into complex of type II is competitive relative to the inhibition reaction. This can partially explain the decrease in the rate of binase inhibition as compared with the corresponding reaction of barnase.